Human papillomaviruses (HPV) are known to be approximately 8 kb DNA viruses and to be closely associated with various malignant tumors, causing uterine cervical cancer to women (Godfroid et al., J. Virol. Method 75:69-81, 1998).
The uterine cervical cancer has been considered to be closely associated with sexual contact, and an HPV infection, which is one of the most common sexually transmitted diseases, is involved in incidences of uterine cervical carcinogenesis. To date, approximately 100 HPV genotypes have been identified; approximately 30 types have been proved to be closely related to cervical cancer, which are subdivided into a “high-risk” HPV type group (e.g. 16, 18, 31, 33, or 35 type) and a “low-risk” HPV type group (e.g. 6, 11, 42, 43, or 44 type) (De Villiers, J. Virol. 63:4898-4903, 1989; Jacobs et al., J. Clin. Microbiol. 33:901-905, 1995.).
The cervical cancer is currently diagnosed by a combination of several diagnostic tests. Among these, the most commonly used for diagnosis of cervical cancer is a Papanicolaou (Pap) smear test. However, the Pap smear test primarily relies on expert's ability, false or inaccurate test results are frequently appeared (Menezes et al., Acta Cytol. 45:919-926, 2001). Colposcopic screening enables HPV infections to be detected relatively accurately, that is, up to 70% of detection rate. However, the colposcopic screening is also problematic because it is incapable of determining HPV genotypes to classify high- and low-risk genotypes. On the other hand, it is a costly procedure and requires a highly-trained expert and expensive equipment (Reid et al., Clin Obstet. Gynecol. 32:157-179, 1989).
A PCR-RFLP method, which uses restriction enzymes after PCR (polymerase chain reaction) amplification of HPV a region of L1 region is accomplished, enables detection results to be obtainable easily and simply. However, according to this method, if variants are not identified by the restriction enzymes used, a highly sensitive assay is not available (Lungu et al., JAMA 267:2493-2496, 1992). In addition, the efficiency of PCR amplification may vary according to HPV genotypes, which may undesirably reduce accuracy of the assay (Qu et al., J. Clin. Microbiol. 35:1304-1310, 1997; Karksen et al., J. Clin. Microbiol. 34:2095-2100, 1996; Gravitt et al., J. Clin. Microbiol. 38:357-361). Further, commercially available hybrid capture kit (Digene, Inc., USA) is identifiable without PCR amplification and they can classify high-risk and low-risk HPV groups. However, the hybrid capture kit cannot distinguish between HPV genotypes 16 and 18, which are highly related with the uterine cervical cancer, and other high-risk HPV genotypes (Clavel et al., J. Clin. Pathol. 51:737-740, 1998). HPV genotype assay kits (BioMedLab., Co., Korea) using HPV DNA chips, which have recently been developed, are subjected to 2-dimensional hybridization on slides, followed by three washing steps, which is quite a burdensome work.
Similarly, HPV detection kits using suspension arrays are currently available. However, the HPV kit exhibits very low signal values for detection, suggesting that there may be limitations in actual detection of low concentrations of infecting viruses, particularly when two or more viruses are co-infected. In addition, to confirm whether there are any other genotypes of HPV without probe on chip or bead or to check a PCR reaction has been carried out properly, the detection technique using micro-array kits requires a number of additional post-PCR steps, such as electrophoresis of PCR products, which are quite burdensome and manageable tasks.
Accordingly, it would be highly desirable to develop high-sensitivity, HPV type detection methods which can accurately detect with extremely low concentrations of HPV types contained in a reagent.
Under these circumstances, the present inventors have tried to detect HPV types with a high sensitivity through the improvement of detection methods especially in the probes for HPV genotype. The present inventors produced detection probes having nucleic acid sequences capable of specifically detecting HPV types from a region of L1 gene with highly heterogeneity between different HPV types. Then, the region was subjected to primary PCR amplification, followed by labeling with a HPV type detection probe to yield a biotin-labeled, single-stranded target molecules. With this labeling methods, the present inventors confirmed that the signal intensity of current labeling methods is approximately 10 times higher than that produced by the conventional method. Further, as described above, the present inventor developed a hybridization probe reacting with all HPV genotypes, enabling even rare HPV infections to be detected without electrophoresis, unlike in the conventional detection probe. In addition, in order to determine whether a PCR reaction has been carried out successfully, GAPDH gene existing in the sample all the time was also amplified and reacted with the hybridization probe for positive control.